Without limiting the scope of the invention, its background is described in connection with classical methods for the identification of microorganisms.
Microorganisms are traditionally identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20E™ system (bioMerieux). For susceptibility testing, clinical microbiology laboratories use methods including disk diffusion, agar dilution and broth microdilution. The detection and identification of biological agents are important in determining the best course of treatment and/or eradication of the biological agent in natural infections, and other cases; such as, but not limited to, biological warfare. Although identifications based on biochemical tests and antibacterial susceptibility tests are cost-effective, generally two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to identify the bacteria from clinical specimens as well as to determine their susceptibility to antimicrobial agents.
There are commercially available automated systems that combine these biochemical identification and susceptibility testing processes; such as, the Mircroscan WalkAway system, the Sensititre ARIS (automatic reading and incubation system) and the Vitek system from bioMerieux, which use sophisticated and expensive apparatus for faster microbial identification and susceptibility testing [1]. These systems require shorter incubation periods, thereby allowing most bacterial identifications and susceptibility testing to be performed in less than 6 hours. Nevertheless, these systems typically require the primary isolation of the bacteria or fungi as a pure culture, a process that takes approximately 18 hours for a pure culture or 48 hours for a mixed culture. Thus, the time from sample reception to identification is at minimum 24 hours. Moreover, it is now accepted that approximately 90% of bacteria, and a large percentage of fungi are fastidious organisms, or do not grow in culture. Identification must rely on labor-intensive techniques such as direct microscopic examination of the specimens and by direct and/or indirect immunological assays. Cultivation of most parasites is impractical in the clinical laboratory. Hence, microscopic examination of the specimen, a few immunological tests and clinical symptoms are often the only methods used for identification; an identification that frequently remains presumptive.
Clinical Specimens Tested in Clinical Microbiology Laboratories. Most clinical specimens received in clinical microbiology laboratories are urine and blood samples. The remaining percentage of clinical specimens comprise various biological fluids including sputum, pus, cerebrospinal fluid, synovial fluid, respiratory tract aspirate, deep pus, ear aspirate, pleural and pericardial fluid, peritoneal fluid, and others. Infections of the urinary tract, the respiratory tract and the bloodstream are usually of bacterial etiology and require antimicrobial therapy. Typically all clinical samples received in a clinical microbiology laboratory are tested routinely for the identification of bacteria and antibiotic susceptibility.
Conventional Pathogen Identification from Clinical Specimens. Urine Specimens. A myriad of tests have been developed to search for pathogens in urine specimens. However, the gold standard remains the classical semi-quantitative plate culture method in which 1 μL of urine is streaked on agar plates and incubated for 18-24 hours. Colonies are then counted to determine the total number of colony forming units (CFU) per liter of urine. A bacterial urinary tract infection (UTI) is normally associated with a bacterial count of 107 CFU/L or more in urine. However, infections with less than 107 CFU/L in urine are possible, particularly in patients with a high incidence of diseases or those catheterized [3]. It is not uncommon for 80% of urine specimens tested in clinical microbiology laboratories are considered negative (i.e. bacterial count of less than 107 CFU/L;). Urine specimens found positive by culture are further characterized using standard biochemical tests to identify the bacterial pathogen and are also tested for susceptibility to antibiotics. The biochemical and susceptibility testing normally require 18-24 hours of incubation.
Accurate and rapid urine screening methods for bacterial pathogens would allow a faster identification of negative specimens and a more efficient treatment and care management of patients. Several rapid identification methods (Uriscreen™, UTIscreen™, Flash Track™ and others) have been compared to standard biochemical methods, which are based on culture of the bacterial pathogens. Although much faster, these rapid tests showed low sensitivities and poor specificities as well as a high number of false negative and false positive results.
Blood Specimens. Blood specimens received in a clinical microbiology laboratory are also submitted for culture. Blood culture systems may be manual, semi-automated or completely automated. The Bactec™ system (from Becton Dickinson) and the Bactalert™ system (from Organon Teklika corporation) are the two most widely used automated blood culture systems. These systems incubate blood culture bottles under optimal conditions for growth of most bacteria. Bacterial growth is monitored continuously to detect early positives by using highly sensitive bacterial growth detectors. Once growth is detected, a gram stain is performed directly from the blood culture and then used to inoculate nutrient agar plates. Subsequently, bacterial identification and susceptibility testing are carried out from isolated bacterial colonies with automated systems as described previously. Blood culture bottles are normally reported as negative if no growth is detected after an incubation of 6 to 7 days. Normally, the vast majority of blood cultures are reported negative.
Other Clinical Samples. Upon receipt by the clinical microbiology laboratory, all body fluids other than blood and urine that are from normally sterile sites (i.e. cerebrospinal, synovial, pleural, pericardial and others) are processed for direct microscopic examination and subsequent culture. Again, most clinical samples are negative for culture. In all these normally sterile sites, tests for the universal detection of algae, archaea, bacteria, fungi and parasites would be very useful.
Regarding clinical specimens that are not from sterile sites such as sputum or stool specimens, the laboratory diagnosis by culture is more problematic because of the contamination by the normal flora. The bacterial or fungal pathogens potentially associated with the infection are grown and separated from the colonizing microbes using selective methods and then identified as described previously. For DNA-based assays for species or genus or family or group detection and identification as well as for the detection of antimicrobial agents' resistance genes from these specimens would be very useful and would offer several advantages over classical identification and susceptibility testing methods.
DNA-Based Assays with any specimen. There is an obvious need for rapid and accurate diagnostic tests for the detection and identification of algae, archaea, bacteria, fungi and parasites directly from clinical specimens. Common diagnostic methods involving DNA sequencing use florescence detected by a camera or laser and/or other optical method for signal detection and measurement. The process of DNA sequencing specifically refers to the determination of the nucleotide order of a particular DNA fragment. DNA-based technologies are rapid and accurate and offer a great potential to improve the diagnosis of infectious diseases [6-8]). The Universal DNA probes and amplification primers which are objects of the present invention for DNA sequencing applications using non-optical base detection methods are applicable for the detection and identification of algae, archaea, bacteria, fungi, and parasites directly from any clinical specimen such as blood, urine, sputum, cerebrospinal fluid, pus, genital and gastro-intestinal tracts, skin or any other type of specimens. These assays are also applicable for the detection and identification, or confirmation of organism identification from microbial cultures (e.g. blood cultures, bacterial or fungal colonies on nutrient agar, or liquid cell cultures in nutrient broth). The DNA based tests proposed in this invention are superior in terms of both speed and accuracy to standard biochemical methods currently used for routine diagnosis from any clinical specimens in microbiology laboratories. Since these tests can be performed in less than 48 hours, they provide the clinician with new diagnostic tools which should contribute to a better management of patients with infectious diseases. Specimens from sources other than humans (e.g. other primates, birds, plants, mammals, farm animals, livestock, food products, environment such as water or soil, and others) may also be tested with these assays.
High Percentage of Culture-Negative Specimens. Among all the clinical specimens received for routine diagnosis, approximately 80% of urine specimens and even more (around 95%) for other types of normally sterile clinical specimens are negative for the presence of bacterial pathogens. It would also be desirable, in addition to identify bacteria at the species or genus or family or group level in a given specimen, to screen out the high proportion of negative clinical specimens with a DNA-based test detecting the presence of any bacterium (i.e., universal bacterial detection). As disclosed in the present invention, such a screening test may be based on DNA amplification by PCR and sequencing of hypervariable regions near a highly conserved genetic target found universally in all bacteria. Specimens negative for bacteria would not be amplified by this assay. On the other hand, those that are positive for any bacterium would give a positive amplification signal, and could be moved forward in the processing pipeline into sequencing analysis for organism identification. Similarly, hyper variable regions of conserved genes of fungi and parasites could serve to map the organisms to its most closely related taxonomic level, and establish the presence of that specific organism in the specimen known to be pathogenic or opportunistic pathogens.
Development of Rapid DNA Sequencing Based Diagnostic Tests. A rapid diagnostic test should have a significant impact on the management of infections. DNA amplification and sequencing technologies offer several advantages over conventional methods for the identification of pathogens and antimicrobial agents resistance genes from clinical samples [6, 9]). There is no need for culture of the pathogens, hence the organisms can be detected directly from clinical samples, thereby reducing the time associated with the isolation and identification of pathogens, and reducing the amount of hazardous biological material that need be disposed. Furthermore, DNA-based sequencing assays are more accurate for microbial identification than currently used phenotypic identification systems which are based on biochemical tests and/or microscopic examination. Commercially available DNA-based sequencing technologies are currently used in clinical microbiology laboratories, mainly for the detection and identification of fastidious bacterial pathogens such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrhoeae as well as for the detection of a variety of viruses [10]. There are also other commercially available DNA-based assays that are used as culture confirmation assays. DNA sequencing based tests for the detection and identification of bacterial pathogens which are detectable by the present invention, for example: Staphylococcus sp. (U.S. Pat. No. 5,437,978), Neisseria sp. (U.S. Pat. No. 5,162,199 and European patent serial no. 0,337,896,131) and Listeria monocytogenes (U.S. Pat. Nos. 5,389,513 and 5,089,386). However, the diagnostic tests described in these patents are based either on rRNA genes or on genetic targets detected by optical detection based sequencing techniques, different from those described in the present invention. To our knowledge there are no other patents published by others describing the use of non-optical based sequencing technology described in the present invention for microbiological diagnostic purposes.
Although there are phenotypic identification methods which have been used for more than 125 years in clinical microbiology laboratories, these methods do not provide information fast enough to be useful in the initial management of patients. There is a need to increase the speed of the diagnosis of commonly encountered bacterial, fungal and parasitical infections. Besides being much faster, DNA-based diagnostic tests are more accurate than standard biochemical tests presently used for diagnosis because the microbial genotype (e.g. DNA level) is more stable than the phenotype (e.g. physiologic level).
Bacteria, fungi and parasites encompass numerous well-known microbial pathogens. Other microorganisms could also be pathogens or associated with human diseases. For example, achlorophylious algae of the Prototheca genus can infect humans. Archaea, especially methanogens, are present in the gut flora of humans [11, 12]. Methanogens have been associated to pathologic manifestations in the colon, vagina, and mouth [11, 13, 14].
In addition to the identification of the infectious agent, it is often desirable to identify harmful toxins and/or to monitor the sensitivity of the microorganism to antimicrobial agents. As presented in this methodology, genetic identification of the microorganism could be performed simultaneously with toxin and antimicrobial agents' resistance genes.
Knowledge of the genomic sequences of algal, archaeal, bacterial, fungal and parasitical species continuously increases as indicated by the number of sequences available from public databases such as GenBank. In order to determine good candidates for diagnostic purposes, one could select sequences for DNA-based assays from genomes available from public databases for (i) the species-specific detection and identification of commonly encountered bacterial, fungal and parasitical pathogens, (ii) the genus-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (iii) the family-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (iv) the group-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (v) the universal detection of algal, archaeal, bacterial, fungal or parasitical pathogens, and/or (vi) the specific detection and identification of antimicrobial agents resistance genes, and/or (vii) the specific detection and identification of bacterial toxin genes. All of the above types of DNA-based assays may be performed directly from any type of clinical specimens or from a microbial culture.
U.S. Pat. No. 6,001,564, and patent publication WO98/20157, teach that DNA sequences described are suitable for: (i) the species-specific detection and identification of clinically important bacterial pathogens, (ii) the universal detection of bacteria, and (iii) the detection of antimicrobial agents resistance genes using amplification, hybridization, and sequencing technology dependent on optical detection systems.
Patent publication WO98/20157 describes proprietary tuf DNA sequences as well as tuf sequences selected from public databases (in both cases, fragments of at least 100 base pairs), as well as oligonucleotide probes and amplification primers derived from these sequences. All the nucleic acid sequences described in that patent publication can be used in: (a) detecting the presence of bacteria and fungi; and (b) detecting specifically at the species, genus, family or group levels, the presence of bacteria and fungi and antimicrobial agents resistance genes associated with these pathogens. However, it is noted that these methods and kits need to be improved, since the ideal kit and method should be capable of diagnosing close to 100% of microbial pathogens and associated antimicrobial agents resistance genes and toxins genes. For example, infections caused by Enterococcus faecium have become a clinical problem because of its resistance to many antibiotics. Both the detection of these bacteria and the evaluation of their resistance profiles are desirable. Non-optical genomic sequencing methods developed for the detection of pathogens in humans and animals fulfill this need by utilizing a non-optical sequencing platform, different than what was originally patented.
Use of highly conserved genes for identification and diagnostics. Highly conserved genes are useful for identification of microorganisms. For bacteria, the most studied genes for identification of microorganisms are the universally conserved ribosomal RNA genes (rRNA). Among those, the principal targets used for identification purposes are the small subunit (SSU) ribosomal 16S rRNA genes (in prokaryotes) and 18S rRNA genes (in eukaryotes) [15, 16]. The rRNA genes are also the most commonly used targets for universal detection of bacteria [17, 18] and fungi [19].
However, it may be difficult to discriminate between closely related species when using primers derived from the 16S rRNA. In some instances, 16S rRNA sequence identity may not be sufficient to guarantee species identity [20], and it has been shown that inter operon sequence variation as well as strain to strain variation could undermine the application of 16S rRNA for identification purposes [21]. The heat shock proteins (HSP) are another family of highly conserved proteins. These ubiquitous proteins in bacteria and eukaryotes are expressed in answer to external stress agents. One of the most described of these HSP is HSP 60. This protein is highly conserved at the amino acid level; hence it has been useful for phylogenetic studies. Similar to 16S rRNA, it would be difficult to discriminate between species using the HSP 60 nucleotide sequences as a diagnostic tool. However, Goh et al. identified a highly conserved region flanking a variable region in HSP 60, which led to the design of universal primers amplifying this variable region (Goh, et al., U.S. Pat. No. 5,708,160). The sequence variations in the resulting amplicons were found useful for the design of species-specific assays.
DNA Sequencing Techniques. In recent years, the overall understanding in biology has been dramatically advanced through the development of fast, sensitive nucleic acid sequencing methods using automated DNA sequencers. DNA sequencing technology is opening many new fields, and is finding novel applications in biology and medicine that go far beyond the initial goal of elucidating the order of nucleotide bases in a molecule of DNA. Nucleic acid sequencing refers to the process of determining the primary structure of an unbranched biopolymer, which results in a symbolic linear depiction know as a ‘sequence’ that summarizes much of the atomic level structure of the sequenced molecule. The process of DNA sequencing specifically refers to the determination of nucleotide order of a particular DNA fragment. It is now possible to analyze entire genomes of bacteria, fungi, viruses, animals, and plants. The major limitations to current sequencing methods are the accuracy of the sequence, the length of an individual fragment (template) that can be sequenced, the cost of the sequence analysis, and the length of time it takes to determine the sequence. Some recent efforts have made significant progress towards the development of methods that improve the ability to prepare genomes for sequencing, and to successfully sequence large numbers of templates simultaneously. The DNA sequencing technologies can be reviewed and considered in a variety of ways. However, for the purposes of this patent, we can separate the technologies fundamentally based on the type of detection method used in the technique to determine nucleotide order. These detection platforms can be separated into Optical and Non-Optical based methods of genome sequencing. Since the inception of genome sequencing in the 1970's until now, Optical genome sequencing techniques have predominated sequencing technology, and are denoted by the requirement for imaging technology, electromagnetic intermediates either in the form of X-rays.
Optical methods of genome sequencing. Maxam-Gilbert Sequencing: The first two sequencing methods were described in 1977. Maxam and Gilbert described a chemical degradation method [26], and Sanger described an enzymatic dideoxy method (also called the chain-terminator method)[22], which became the method of choice since it was perceived to be more efficient and use fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert. Maxam and Gilbert's method requires radioactive labeling at one 5′ end of the DNA, typically by a kinase reaction using gamma-32P ATP, and purification of the DNA fragment to be sequenced. The fragments are visualized by exposing the gel with the separated fragments to X-ray film, presenting a series of bands that each correspond to a labeled DNA fragment. From these fragments, the DNA sequence could be inferred.
Sanger Sequencing: The Sanger method uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators to generate a set of nucleic acid fragments which are different in length by one nucleotide. Each one of these chain terminating dideoxynucleotides (e.g. ddATP, ddGTP, ddCTP, and ddTTP) can be uniquely labeled. The labeled DNA fragments are size separated by gel electrophoresis with single nucleotide resolution. Variations in the electrophoretic process include applications of slab gels, capillaries, or microfluidic devices using denaturing polyacrylamide-urea gels, or other gradient poor-size polymer matrices. The DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. Different variations of chain-termination sequencing have included tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled with a fluorescent dyes. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. Thus, these fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
Dye-terminator sequencing: Dye-terminator sequencing is differentiated by labeling the chain terminator ddNTPs each with a different and unique fluorescent dye that emits light at a unique wavelength. This permits sequencing in a single reaction, rather than four reactions as in the labeled-primer method. Even though the Sanger sequencing was the only method utilized in the parallel consortia that determined the complete human genome, many limitations of the Sanger processed were realized; such as, the need for gels or polymers used as sieving separation media for the fluorescently labeled DNA fragments, the low number of samples which could be analyzed in parallel, and the difficulty of total automation of the sample preparation methods. These limitations shifted focus to develop techniques without gels allowing sequence determination on very large numbers of samples in parallel.
454 Genome Sequencer FLX instrument made by Roche Applied Science. The first ‘next generation’ sequencing system on the market was developed by 454 Life Sciences and introduced in 2005. Within this instrument, DNA fragments are ligated with specific adapters that cause the binding of one fragment to a bead. Emulsion PCR is carried out for fragment amplification, with water droplets containing one-bead and PCR reagents immersed in oil. Amplification is needed to obtain sufficient light signal intensity for reliable detection in the so-called ‘sequencing-by-synthesis’ reaction steps. When PCR amplification cycles are completed and after denaturation, an individual bead with a single amplified fragment is placed at the top end of an etched fiber in an optical fiber chip, created from a glass fiber bundle. Each glass fiber serves as optical waveguide, which transfers light to its other end attached to a CCD camera, enabling positional detection of emitted light. Therefore, each bead has an addressable position in the light guide chip, containing hundreds of thousands of available positions. In a subsequent step, polymerase enzyme and primer are added to each of the beads, along with one unlabeled nucleotide per bead, thus starting the synthesis of the complementary strand. The incorporation of the following base by the polymerase enzyme in the growing chain releases a pyrophosphate group, which is then detected as emitted light. This method has achieved DNA read length to the 400-500 base range, with paired end reads. Drawbacks to this method are a relatively high cost of operation and generally lower reading accuracy in homopolymer stretches of identical bases and generally lower reading accuracy in homopolymer stretches of identical bases.
Illumina (Solexa) Genome Analyzer: The Solexa sequencing platform was first commercialized in 2006, and was acquired by Illumina in 2007. The functioning principles of this instrument are based on the same sequencing-by-synthesis chemistry. DNA fragments are ligated at both ends to adapters and, after denaturation, immobilized at one end on a solid support. The surface of the support is coated densely with the adapters and the complementary adapters. Each single-stranded fragment, immobilized at one end on the surface, creates a ‘bridge’ structure by hybridizing with its free end to the complementary adapter on the surface of the support. In the mixture containing the PCR amplification reagents, the adapters on the surface act as primers for the PCR amplification. PCR amplification is needed as a step in this system as well to ensure sufficient light signal intensity for reliable detection of added bases. The PCR step creates clusters of single-stranded DNA fragments on the surface of the support called ‘polonies’. The novelty of this system occurs in the next step following amplification, where the reaction mixture for the sequencing reactions and DNA synthesis is supplied onto the surface and contains primers, four reversible terminator nucleotides each labeled with a different fluorescent dye and the DNA polymerase. After incorporation into the DNA strand, the terminator nucleotide, as well as its position on the support surface, is detected and identified by its fluorescent dye at the CCD camera. This system achieved sequence read lengths of approximately 35 nucleotides, and the sequence of 40 million polonies can be simultaneously determined in parallel. Updates to the Illumina system have been the introduction of a paired-end module, new optics and camera components that allowed the system to triple the output per paired-end run from 1 to 3 Gb of data [32].
Applied Biosystems ABI SOLiD system: ABI introduced the SOLiD system in 2007 uses ligation chemistry as its primary platform. In this technique, DNA fragments are ligated to adapters then bound to beads. A water droplet in oil emulsion contains the amplification reagents and only one fragment bound per bead; DNA fragments on the beads are amplified by emulsion PCR. Once amplified, the DNA are denatured After DNA, and the beads are deposited onto a glass support surface. In the next steps, a primer is hybridized to the adapter, followed by the hybridization of a mixture of oligonucleotide octamers followed by the addition of the ligation mixture. Utilizing four unique fluorescent labels and repeated series of hybridization and ligations cycles, the DNA sequence is determined by interrogating every 1st and 2nd base in each ligation reaction. Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length of the DNA strand. The sequencing process may be continued in the same way with another primer shorter by one base than the previous one, and in fact is done so five times. Through this primer ‘reset’ process, theoretically every base is interrogated in two independent ligation reactions by two different primers. Thus, the sequence read length is shorter, respectively speaking, at about 35 bases. However, the method has proven to be very accurate as a result of this dual interrogation type format.
Non-Optical Methods of Genome Sequencing: The previously outlined optical based methods are still hindered by relatively large reaction volume size needed to prepare templates that are detectable by theses systems, the need for special nucleotide analogues as reagents, and complicated enzymatic and/or chemiluminescence reactions to generate detectable optical signals. As a result of these limitations, a major shift towards non-optical based sequencing methods occurred, resulting in the development of sequencing techniques with two other major categories of detection schemes; sequencing based mass spectrometry, and sequencing based on integrated circuits.
Nucleic Acid Sequencing based on Mass Spectrometry: In U.S. Pat. No. 7,501,251, methods are described for detecting a target nucleic acid in a biological sample using RNA amplification using a primer comprising a sequence that is complementary to a polynucleotide sequence in the target nucleic acid, and a sequence that encodes an RNA polymerase promoter. The RNA polymerase that recognizes the promoter is used to synthesize RNA. The newly synthesized RNA is detected by mass spectrometry, which establishes the presence or absence of that target RNA in the biological sample. The detection systems of mass spectrometers provide a means of determining the individual mass and charge of volatilized molecules in a vacuum as the trajectory of the ‘flying’ molecule is influenced by combinations of electric and magnetic fields. This technique is an example of MS-based proteomics; a discipline made possible by the availability of gene and genome sequence databases and technical and conceptual advances primarily in the area of protein ionization methods.
Nucleic Acid Sequencing using integrated semiconductor devices. Recent advances in the field of photonic imaging have produced very large, fast arrays of electronic sensors. This technology was adapted for the construction of an integrated circuit to detect the hydrogen ions that would be released by NNA polymerase during sequencing by synthesis rather than a sensor designed for the detection of a photon[34]. The Ion Torrent was developed by Life Technologies using the ion-sensitive field-effect transistor (ISFET) due to its sensitivity to hydrogen ions and compatibility with CMOS processes [35]. The Ion Torrent was not the first effort to detect both single-nucleotide polymorphisms [36], monitor DNA synthesis [37], or electronically sequence DNA[38]. None of these earlier attempts were able to produce de novo DNA sequence, address issues of delivering template NDA to the sensors, or scale the entire system to large arrays [35]. Prior to the Ion Torrent, ISFETs were limited in the number of sensors per array, the yield of working independent sensors and readout speed [39], and had issues protecting the electronic circuitry from fluid once the sensors were exposed [40]. With this new technology, 25 million bases can be generated from chips containing 1.2 million sensors. This capability was demonstrated in by Rothberg et. al [35].
U.S. Pat. No. 7,948,015 focuses on the development of the ion sensor chips, supporting instrumentation, and software to enable de novo DNA sequencing for applications requiring millions to billions of bases. The method described here will utilize universal or broad range primers and individual sample specific barcodes or tags as have been well described in the literature, in order to analyzed batches or multiple specific specimens or subjects or sample detecting many specific targets that are grouped together to create a single assay. This assay will be analyzed using a cost effective semi-conductor technology or other non-optical method for determining the sequence of molecular material such as proteins or nucleic acids (RNA or DNA). One example is to sequence each of the multiplexed analytes based upon pH generation detected using a semi-conductor or other chip-based technology. This allows many analytes to be screened all at once using broad range (e.g. kingdom specific, genus specific, family or class or sub-groups of organisms or targets) to be screened all at once and allows many different samples to be analyzed all together.